Vegetative insecticidal proteins useful for control of insect pests

ABSTRACT

DIG-657 vegetative insecticidal toxins, polynucleotides encoding such toxins, use of such toxins to control pests, and transgenic plants that produce such toxins are disclosed. The invention includes DIG-657 variants, fragments and analogs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Non-Provisional applicationSer. No. 14/740,326 filed Jun. 16, 2016, which claims priority from andthe benefit of U.S. Provisional Application 62/014,916, filed Jun. 20,2014, and U.S. Non-Provisional application Ser. No. 14/740,326, filedJun. 16, 2015. The entire contents of these applications are herebyincorporated by reference into this application.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“74948-US-DIV_20171128_Seq_Listing_DIG17_ST25”, created on Nov. 28,2017, and having a size of 53 kilobytes, and is filed concurrently withthe specification. The sequence listing contained in this ASCIIformatted document is part of the specification, and is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

This invention generally relates to the field of molecular biology. Morespecifically the invention concerns new insecticidal protein toxinsdeveloped from a new vegetative insecticidal protein toxin found inBacillus thuringiensis and their use to control insects.

BACKGROUND OF INVENTION

Insects and other pests cost farmers billions of dollars annually incrop losses and expense to keep these pests under control. In additionto losses in field crops, insect pests are also a burden to vegetableand fruit growers, to producers of ornamental flowers, and to homegardeners. The losses caused by insect pests in agricultural productionenvironments include decrease in crop yield, reduced crop quality, andincreased harvesting costs.

Insect pests are mainly controlled by intensive applications of chemicalpesticides, which are active through inhibition of insect growth,prevention of insect feeding or reproduction, or cause death. Goodinsect control can thus be reached, but these chemicals can sometimesalso affect other beneficial insects. Another problem resulting from thewide use of chemical pesticides is the appearance of resistant insectpopulations. This has been partially alleviated by various resistancemanagement practices, but there is an increasing need for alternativepest control agents. Biological pest control agents, such as Bacillusthuringiensis (Bt) strains expressing pesticidal toxins likedelta-endotoxins, have also been applied to crop plants withsatisfactory results, offering an alternative or compliment to chemicalpesticides. The genes coding for some of these delta-endotoxins havebeen isolated and their expression in heterologous hosts have been shownto provide another tool for the control of economically important insectpests. In particular, the expression of insecticidal toxins, such asBacillus thuringiensis delta-endotoxins, in transgenic plants haveprovided efficient protection against selected insect pests, andtransgenic plants expressing such toxins have been commercialized,allowing farmers to reduce applications of chemical insect controlagents.

The soil microbe Bacillus thuringiensis is a Gram-positive,spore-forming bacterium characterized by parasporal crystalline proteininclusions. Bacillus thuringiensis continues to be the leading source ofnovel insecticidal proteins for development of plant incorporatedpesticides. Using various strains of bacterial isolates, we haveinvented new Bt toxins that are active against commercially importantinsect pests. In the North American maize insect resistance market,Spodoptera frugiperda (fall armyworm “FAW”), Ostrinia nubialis Hubner(European corn borer “ECB”), and Helicoverpa zea Boddie (corn earworm“CEW”) are the key driver pests, although there are other key insectpests in other geographies (e.g. Helicoverpa armigera (cotton bollworm“CBW” or corn earworm “CEW”)) and additional secondary, but importantinsect pest species. Bt toxins represent over 90% of the bioinsecticidemarket and essentially the entire source of genes for transgenic cropsthat have been developed to provide resistance to insect feeding. Btbacteria produce insecticidal delta-endotoxins including Crystal (Cry),Cytotoxin (Cyt), and Vegetative Insecticidal Protein (VIP) toxins,depending on their gene and protein structure. Cry toxins are producedduring spore formation as insoluble crystal proteins. VIP toxins, on theother hand, are produced as soluble proteins during the vegetative stageof Bt bacterial growth. VIP proteins are distinct from Cry proteins intheir structure, but share the property with Cry toxins of being poreformers acting on cells located in the insect midgut (Yu, C.-G., et al.,1997 Appl. Environ. Microbiol. 63:532-536, Lee, M. K., et al., 2003,Appl. Environ. Microbiol. 69: 4648-4657, Shotkoski, F., et al., 2003,Proc. Beltwide Cotton Conf, 89-93). We describe here the invention ofnew VIP toxins that have broad spectrum insecticidal activity, includinginsecticidal activity against ECB, which is unique compared to VIPproteins currently known (Yu, C.-G., et al., 1997 Appl. Environ.Microbiol. 63:532-536).

Patent documents WO2013/134523, WO 94/21795, WO 96/10083, U.S. Pat. Nos.5,877,012, 6,107,279, 6,137,033, and 6,291,156, as well as Estruch etal. (1996, Proc. Natl. Acad. Sci. 93:5389-5394) and Yu et al. (1997,Appl. Environ. Microbiol. 63:532-536), describe a class of insecticidalproteins called VIP3. VIP3 genes encode approximately 88 kDa proteinsthat are produced and secreted by Bacillus during its vegetative stagesof growth. These toxins were reported to be distinct fromcrystal-forming delta-endotoxins. These documents make specificreference to toxins designated VIP1A(a), VIP1A(b), VIP2A(a), VIP2A(b),VIP3A(a), and VIP3A(b). See also Lee et al., AEM vol. 69, no. 8 (August2003), pages 4648-4657, for a discussion of the mechanism of action andtruncation of VIP proteins.

The VIP3A protein possesses insecticidal activity against a widespectrum of lepidopteran pests, including FAW, CEW, Agrotis ipsilonHufnagel (black cutworm “BCW”), and Heliothis virescens Fabricius(tobacco budworm “TBW”). More recently, VIP proteins have been found tobe toxic to certain species of hemipteran insect pests (Nanasaheb, P. etal, Toxins (Basel) vol. 4, no. 6 (June 2012), pages 405-429, Sattar S.and Maiti M. K., J. Microbiol. Biotechnol. 2011, 21:937-946). Thus, theVIP class of proteins display a unique spectrum of insecticidalactivities. Other disclosures, WO 98/18932, WO 98/33991, WO 98/00546,and WO 99/57282, have also now identified homologues of the VIP3 classof proteins.

The continued use of chemical and biological agents to control insectpests heightens the chance for insects to develop resistance to suchcontrol measures. Also, the high selectivity of biological controlagents often results in only a few specific insect pests beingcontrolled by each agent. Despite the success of ECB-resistanttransgenic corn, the possibility of the development of resistant insectpopulations threatens the long-term durability of Cry proteins in ECBcontrol and creates the need to discover and develop new Cry or othertypes of biological control agents to control ECB and other pests.Insect resistance to Bt Cry proteins can develop through severalmechanisms (Heckel et al., 2007, Pigott and Ellar, 2007). Multiplereceptor protein classes for Cry proteins have been identified withininsects, and multiple examples exist within each receptor class.Resistance to a particular Cry protein may develop, for example, bymeans of a mutation within the toxin-binding portion of a cadherindomain of a receptor protein. A further means of resistance may bemediated through a protoxin-processing protease. Thus, resistance to Crytoxins in species of Lepidoptera has a complex genetic basis, with atleast four distinct, major resistance genes. Lepidopteran insectsresistant to Cry proteins have developed in the field within the speciesDBM (diamondback moth) (Tabashnik, 1994), Trichoplusia ni Hubner(cabbage looper “CL”; Janmaat and Myers 2003, 2005), and CEW (Tabashniket al., 2008). Therefore development and deployment of new high potencyplant incorporated pesticidal proteins such as those disclosed hereinare both useful and needed.

Therefore, there remains a need to discover new and effective pestcontrol agents that provide an economic benefit to farmers and that areenvironmentally acceptable. Particularly needed are control agentstargeted to a wide spectrum of economically important insect pests thatefficiently control insect populations that are, or could become,resistant to existing insect control agents and those with equal to orincreased potency compared to current control agents.

BRIEF SUMMARY OF THE INVENTION

The present invention provides insecticidal VIP toxins, including theprotein toxin designated herein as DIG-657 as well as variants ofDIG-657, nucleic acids encoding these toxins, methods of controllingpests using the toxins, methods of producing the toxins in transgenichost cells, and transgenic plants that express the toxins. The inventionfurther provides nucleic acid constructs comprising a nucleic acidsequence encoding an insecticidal protein selected from the groupconsisting of DIG-657, a DIG-657 variant, and a DIG-657 fragment.Isolated insecticidal proteins selected from the group consisting ofDIG-657, a DIG-657 variant, a DIG-657 fragment and an insecticidalprotein comprising residues 206 to 803 of SEQ ID NO:2 are disclosed.Also disclosed are plants, plant parts, and seeds comprising a nucleicacid sequence encoding a protein selected from the group consisting ofDIG-657, a DIG-657 variant, and a DIG-657 fragment. A method forcontrolling an insect pest population comprising contacting individualsin said pest population with a pesticidally effective amount of apolypeptide comprising residues 206 to 803 of SEQ ID NO:2 is alsodisclosed.

In one embodiment the invention provides an isolated DIG-657 insecttoxin polypeptide comprising a core toxin segment selected from thegroup consisting of (a) a polypeptide comprising the amino acid sequenceof residues 206 to 803 of SEQ ID NO:2; (b) a polypeptide comprising anamino acid sequence having at least 90% sequence identity to the aminoacid sequence of residues 206 to 803 of SEQ ID NO:2; (c) a polypeptidecomprising an amino acid sequence of residues 206 to 803 of SEQ ID NO:2with up to 20 amino acid substitutions, deletions, or modifications thatdo not adversely affect expression or activity of the toxin encoded bySEQ ID NO:2; or an insecticidally active fragment thereof.

In another embodiment the invention provides an isolated DIG-657 insecttoxin polypeptide comprising a DIG-657 core toxin segment selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence of residues 1 to 803 of SEQ ID NO:2; (b) a polypeptidecomprising an amino acid sequence having at least 95% or 96% or 97% or98% or 99% sequence identity to the amino acid sequence of residues 1 to803 of SEQ ID NO:2; (c) a polypeptide comprising an amino acid sequenceof residues 1 to 803 of SEQ ID NO:2 with up to 20 amino acidsubstitutions, deletions, or modifications that do not adversely affectexpression or activity of the toxin encoded by SEQ ID NO:1; or aninsecticidally active fragment thereof; (d) a polypeptide comprising anamino acid sequence having at least 93% or 94% or 95% or 96% or 97% or98% or 99% sequence identity to the amino acid sequence of residues 206to 803 of SEQ ID NO:2 that do not adversely affect expression or toxinactivity.

In another embodiment the invention provides fertile plants comprising aDIG-657 insect toxin. The present invention further provides a method ofproducing an insect-resistant or insect tolerant transgenic plant,comprising introducing a nucleic acid molecule of the invention into thetransgenic plant, wherein the nucleic acid molecule is expressible inthe transgenic plant in an effective amount to control insects.

In another embodiment the invention provides a method for controlling apest population comprising contacting said population with apesticidally effective amount of a DIG-657 insect toxin.

In another embodiment the invention provides isolated nucleic acidmolecules that encode a DIG-657 toxin of the invention. Given an aminoacid sequences for DIG-657 toxins, coding sequences can be designed byreverse translating the amino acid sequence using codons preferred bythe intended host plant, and then refining sequences using alternativecodons to remove sequences that might cause problems and provideperiodic stop codons to eliminate long open coding sequences in thenon-coding reading frames.

In another embodiment the invention provides DNA constructs comprising anucleotide sequence that encodes a DIG-657 insect toxin operably linkedto a promoter that is not derived from Bt and is capable of drivingexpression in a plant. The invention also provides a transgenic plantthat comprises the DNA construct stably incorporated into its genome anda method for protecting a plant from a pest comprising introducing theconstruct into said plant.

In yet another embodiment, the invention provides a method for producingan insect resistant or insect tolerant plant comprising breeding a nontransgenic plant with a transgenic plant comprising a foreign DNAconstruct, capable of expressing a DIG-657 toxin, stably incorporatedinto the genome of the plant and selecting progeny by analyzing for atleast a portion of the foreign DNA construct emanating from thetransgenic plant.

DESCRIPTION OF THE DRAWING

FIG. 1 is an image of an SDS-PAGE of purified DIG-657. Lane 1, molecularweight marker; lane 2, 3 μg purified full length DIG-657; lane 3,reaction products of DIG-657 after the purified full length protein istreated with trypsin (1:10 DIG-657 wt/trypsin wt) for 1 hr.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 DNA sequence encoding full-length DIG-657 insect toxin.

SEQ ID NO:2 The deduced DIG-657 protein sequence.

SEQ ID NO:3 Maize-optimized DNA sequence encoding DIG-657 variant 1.

SEQ ID NO:4 DIG-657 variant 2 with codons optimized for expression inPseudomonas fluorescens.

SEQ ID NO:5 DIG-657 v4, Maize optimized High GC content.

SEQ ID NO:6 DIG-657 v5 Maize optimized High GC content of DIG-657truncated 205 AA from the N-terminal.

SEQ ID NO:7 The deduced protein sequence of SEQ ID NO:6.

SEQ ID NO:8 The full length DIG-657 soybean most preferred codonoptimized version.

SEQ ID NO:9 The soybean most preferred codon optimized version oftruncated DIG-657.

SEQ ID NO:10 DNA encoding chloroplast transit peptide 4 (Trap4) fused tothe soybean most preferred codon optimized version of full lengthDIG-657.

SEQ ID NO:11 Deduced protein sequence of TraP4 DIG-657 full lengthsoybean most preferred codon.

SEQ ID NO:12 Trap 4 fused to the soybean most preferred codon optimizedversion of truncated DIG-657.

SEQ ID NO:13 Deduced protein sequence of Trap4 DIG-657 truncatedversion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides insecticidal protein toxins and methodsfor delivering the toxins that are functionally active and effectiveagainst many orders of insects, preferably Lepidopteran insects. By“functional activity” (or “active against”) it is meant that theproteins function as orally active toxin or insect control agents, thatthe proteins have a toxic effect, or are able to disrupt or deter insectgrowth or feeding. When an insect comes into contact with an effectiveamount of a toxin of the subject invention delivered via transgenicplant expression, formulated protein composition(s), sprayable proteincomposition(s), a bait matrix or other delivery system, the results aretypically death of the insect, inhibition of the growth or proliferationof the insect, or prevention of the insects from feeding upon thesource, preferably a transgenic plant, that makes the toxins availableto the insects. Functional proteins of the subject invention can alsowork together or alone to enhance or improve the activity of one or moreother toxin proteins. The terms “toxic,” “toxicity,” or “toxin” aremeant to convey that the subject toxins have functional activity asdefined herein.

Complete lethality to feeding insects is preferred but is not requiredto achieve functional activity. If an insect avoids the toxin or ceasesfeeding, that avoidance will be useful in some applications, even if theeffects are sublethal or lethality is delayed or indirect. For example,if insect resistant transgenic plants are desired, the reluctance ofinsects to feed on the plants is as useful as lethal toxicity to theinsects because the ultimate objective is avoiding insect-induced plantdamage.

A nucleic acid encoding DIG-657 was discovered and isolated from Btstrain PS46L. By “isolated” applicants mean that the nucleic acidmolecules have been removed from their native environment and have beenplaced in a different environment by the hand of man. Because of theredundancy of the genetic code, a variety of different DNA sequences canencode the amino acid sequences disclosed herein. It is well within theskill of a person trained in the art to create these alternative DNAsequences encoding the same, or essentially the same, toxins once theamino acid sequences are known. The nucleic acid sequence for the fulllength coding region of DIG-657 (SEQ ID NO:1) was determined, and thefull length protein sequence of DIG-657 (SEQ ID NO:2) was deduced fromthe nucleic acid sequence. The DIG-657 protein sequence was queriedagainst the GenomeQuest databases “GQ-Pat Platinum protein” and “GQ-PatGoldPlus protein”, as well as the GenBank non-redundant proteindatabase. The closest known homologs are XMI335 (94% identity,WO2013134523-002) and Vip3Ba (74% identity, Accession No. AAV70653, Ranget al).

Insect active variants of the DIG-657 toxin are also described herein,and are referred to collectively as DIG-657 insect toxins, or variantsand include fragments and truncated forms. Individual variants ofDIG-657 may be identified by specific DIG-nomenclature. DIG-657 toxinsare ideal candidates for use to control Lepidopteran pests.

A surprising property of DIG-657 and its variant toxins is that theywere found to be active against populations of ECB and DBM that areresistant to Cry1F and Cry1A toxins. Accordingly, DIG-657 toxins areideal candidates for control and prevention of resistant Lepidopteranpest populations.

The DIG-657 toxins of the invention are active against Lepidopteraninsects, preferably against DBM, ECB, FAW, CEW, CBW, and TBW.Insecticidal activity is also expected for BCW, CL, Spodoptera exigua(beet armyworm “BAW”), Pectinophora gossypiella (pink bollworm),Cochyles hospes Walsingham (banded sunflower moth), and Homoeosomaelectellum (sunflower head moth).

Insecticidal activity of DIG-657 produced in Pseudomonas fluorescens wasdemonstrated to be active on Lepidopteran species including ECB;cry1F-resistant ECB (rECB), DBM, cry1A-resistant DBM (rDBM), CEW, BCW,and TBW. DIG-657 protein was also tested for activity on CBW, FAW andCry1F-resistant FAW (rFAW).

This spectrum of biological activity against both insect pests of maizeand soybean is highly advantageous. The trypsin truncated toxin (SEQ IDNO:7) was tested against ECB in bioassay and shown to be approximatelyequal in activity to the full length DIG-657, indicating that the first205 amino acids on the N-terminus of the protein are not required forbiological activity.

Full length DIG-657 has an intact N-terminus and when treated withtrypsin (1:10 trypsin/DIG-657), the protein is cleaved to two bands, oneat approximately 65 kDa and another one at about 18 kDa (FIG. 1). Inaddition, a small residual full length DIG-657 was also present.N-terminal amino acid analysis of the 65 kDa cleaved product of DIG-657indicated that the site of trypsin cleavage is (205K/S206).

Nucleotide sequences that encode DIG-657, its variants, truncations andfragments, may be synthesized and cloned into standard plasmid vectorsby conventional means, or may be obtained by standard molecular biologymanipulation of other constructs containing the nucleotide sequences.Unique restriction sites internal to a DIG-657 coding region may beidentified and DNA fragments comprising the sequences between therestriction sites of the DIG-657 coding region may be synthesized, eachsuch fragment encoding a specific deletion, insertion or other DIG-657variation. The DNA fragments encoding the modified DIG-657 fragments maybe joined to other DIG-657 coding region fragments or other Cry or VIPcoding region fragments at appropriate restriction sites to obtain acoding region encoding the desired full-length DIG-657 protein, deletedor variant DIG-657 protein. For example, one may identify an appropriaterestriction recognition site at the start of a first DIG-657 codingregion, and a second restriction site internal to the DIG-657 codingregion. Cleavage of this first DIG-657 coding region at theserestriction sites would generate a DNA fragment comprising part of thefirst DIG-657 coding region. A second DNA fragment flanked byanalogously-situated compatible restriction sites specific for anotherDIG-657 coding region or other VIP3 coding region may be used incombination with the first DNA restriction fragment to construct avariant.

Anti-toxin antibodies. Antibodies to the toxins disclosed herein, orfragments of these toxins, can be prepared using standard procedureswell known in the art. Such antibodies are useful to detect the presenceof DIG-657 toxins in plant tissues and a variety of other substances.Such antibodies and anti-sera are useful in various methods of detectingthe claimed DIG-657 toxins of the invention, and variants or fragmentsthereof. It is well known that antibodies labeled with a reporting groupcan be used to identify the presence of antigens in a variety ofmilieus. Antibodies labeled with radioisotopes have been used inradioimmuno assays to identify, with great precision and sensitivity,the presence of antigens in a variety of biological fluids. Morerecently, enzyme labeled antibodies have been used as a substitute forradiolabeled antibodies in the ELISA assay. Further, antibodiesimmunoreactive to the Bt insecticidal toxin of the present invention canbe bound to an immobilizing substance such as a polystyrene well orparticle and used in immunoassays to determine whether the Bt toxin ispresent in a test sample. Anti-DIG-657 antibodies are also used forisolating quantities of DIG-657 toxins from recombinant productionsystems or natural sources.

Transgenic Expression of DIG-657. The subject protein toxins can be“applied” or provided to contact the target insects in a variety ofways. For example, DIG-657 toxins can be used as plant-incorporatedprotectants in transgenic plants (produced by and present in the plant)and are well-known in the art. Expression of the toxin genes can alsoachieve selectivity in specific tissues of the plants, such as theroots, leaves, etc. This can be accomplished via the use oftissue-specific promoters well known in the art.

A preferred embodiment of the subject invention is the transformation ofplants with genes encoding the subject insecticidal protein or itsvariants. The transformed plants are resistant to attack by an insecttarget pest by virtue of the presence of controlling amounts of thesubject insecticidal protein or its variants in the cells of thetransformed plant. By incorporating and expressing genetic material thatencodes a DIG-657 toxin into the genome of a plant eaten by a particularinsect pest, the adult or larvae will die after consuming the foodplant. Numerous members of the monocotyledonous and dicotyledonousclassifications have been transformed. Transgenic agronomic crops aswell as fruits and vegetables are of commercial interest. Such cropsinclude, but are not limited to, maize, rice, soybeans, canola,sunflower, alfalfa, sorghum, wheat, cotton, peanuts, tomatoes, potatoes,and the like. Numerous well known techniques exist for introducingforeign genetic material into monocot or dicot plant cells, and forobtaining fertile plants that stably maintain and express the introducedgene.

In one preferred embodiment, DIG-657 or a variant is delivered orallythrough a transgenic plant comprising a nucleic acid sequence thatexpresses a toxin of the present invention. The present inventionprovides a method of producing an insect-resistant transgenic plant,comprising introducing a nucleic acid molecule of the invention into theplant wherein the toxin is expressible in the transgenic plant in aneffective amount to control an insect. In a non-limiting example, abasic cloning strategy may be to subclone full length or modifiedDIG-657 coding sequences into a plant expression plasmid at Nco1 andSac1 restriction sites. The resulting plant expression cassettescontaining the appropriate DIG-657 coding region under the control ofplant expression elements, (e.g., plant expressible promoters, 3′terminal transcription termination and polyadenylate additiondeterminants, and the like) are subcloned into a binary vector plasmid,utilizing, for example, Gateway® technology or standard restrictionenzyme fragment cloning procedures. LR Clonase™ (Invitrogen, Carlsbad,Calif.) for example, may be used to recombine the full length andmodified gene plant expression cassettes into a binary planttransformation plasmid if the Gateway® technology is utilized. It isconvenient to employ a binary plant transformation vector that harbors abacterial gene that confers resistance to the antibiotic spectinomycinwhen the plasmid is present in E. coli and Agrobacterium cells. It isalso convenient to employ a binary vector plasmid that contains aplant-expressible selectable marker gene that is functional in thedesired host plants. Examples of plant-expressible selectable markergenes include but are not limited to those that encode theaminoglycoside phosphotransferase gene (aphII) of transposon Tn5, whichconfers resistance to the antibiotics kanamycin, neomycin and G418, aswell as those genes which code for resistance or tolerance toglyphosate; hygromycin; methotrexate; phosphinothricin (bialaphos),imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such aschlorosulfuron, bromoxynil, dalapon and the like.

Alternatively, the plasmid structure of the binary plant transformationvector containing the DIG-657 gene insert is performed by restrictiondigest fingerprint mapping of plasmid DNA prepared from candidateAgrobacterium isolates by standard molecular biology methods well knownto those skilled in the art of Agrobacterium manipulation.

Those skilled in the art of obtaining transformed plants viaAgrobacterium-mediated transformation methods will understand that otherAgrobacterium strains besides Z707S may be used, and the choice ofstrain may depend upon the identity of the host plant species to betransformed.

Insect Bioassays of transgenic Arabidopsis. Transgenic Arabidopsis linesexpressing modified DIG-657 proteins can be used to demonstrate activityagainst sensitive insect species in artificial diet overlay assays.Protein extracted from transgenic and non-transgenic Arabidopsis linesmay be quantified by appropriate methods and sample volumes adjusted tonormalize protein concentration. Bioassays are then conducted onartificial diet as described below. Non-transgenic Arabidopsis and/orbuffer and water should be included in assays as background checktreatments.

Bioassay of transgenic maize. Bioactivity of the DIG-657 toxins andvariants produced in plant cells also may be demonstrated byconventional bioassay methods (see, for example Huang et al., 2006).Efficacy may be tested by feeding various plant tissues or tissue piecesderived from a plant producing a DIG-657 toxin to target insects in acontrolled feeding environment. Alternatively, protein extracts may beprepared from various plant tissues derived from a plant producing theDIG-657 toxin and incorporate the extracted proteins in an artificialdiet bioassay. It is to be understood that the results of such feedingassays are to be compared to similarly conducted bioassays that employappropriate control tissues from host plants that do not produce theDIG-657 protein or variants, or to other control samples.

DIG-657 toxins, and insecticidally active variants. In addition to thefull length DIG-657 toxin of SEQ ID NO:2, the invention encompassesinsecticidally active variants of SEQ ID NO:2, SEQ ID NO:11, or SEQ IDNO:13. By the term variant, applicants intend to include certaindeletion, substitution, and insertion mutants. A DIG-657 fragment is anyprotein sequence that is found in SEQ ID NO:2 that is less than the fulllength DIG-657 amino acid sequence and which has insecticidalproperties. DIG-657 variant fragments are also contemplated as part ofthe invention and are defined as fragments of DIG-657 containing certaindeletion, substitution, and insertion mutants described herein and whichhave insecticidal activity. As a preface to describing variants of theDIG-657 toxins that are included in the invention, it will be useful tobriefly review the architecture of DIG-657 protein toxins.

DIG-657 variants created by making a limited number of amino aciddeletions, substitutions, or additions. Amino acid deletions,substitutions, and additions to the amino acid sequence of SEQ ID NO:2can readily be made in a sequential manner and the effects of suchvariations on insecticidal activity can be tested by bioassay. Providedthe number of changes is limited in number, such testing does notinvolve unreasonable experimentation. The invention also includesinsecticidally active variants of the core toxin segment (amino acids206-803 of SEQ ID NO:2, or in which up to 10, up to 15, or up to 20independent amino acid additions, deletions, or substitutions have beenmade).

Variants may be made by making random mutations or the variants may bedesigned. In the case of designed mutants, there is a high probabilityof generating variants with similar activity to the native toxin whenamino acid identity is maintained in critical regions of the toxin whichaccount for biological activity or are involved in the determination ofthree-dimensional configuration which ultimately is responsible for thebiological activity. A high probability of retaining activity will alsooccur if substitutions are conservative. Amino acids may be placed inthe following classes: non-polar, uncharged polar, basic, and acidic.Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type are least likely tomaterially alter the biological activity of the variant. Table 1provides a listing of examples of amino acids belonging to each class.

TABLE 1 Class of Amino Acid Examples of Amino Acids Nonpolar Side ChainsAla (A), Val (V), Leu (L), Ile (I), Pro (P), Met (M), Phe (F), Trp (W)Uncharged Polar Side Chains Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),Asn (N), Gln (Q) Acidic Side Chains Asp (D), Glu (E) Basic Side ChainsLys (K), Arg (R), His (H) Beta-branched Side Chains Thr, Val, IleAromatic Side Chains Tyr, Phe, Trp, His

Pesticidal proteins of the present invention are encoded by a nucleotidesequence sufficiently identical to the nucleotide sequence of SEQ IDNO:1, or the pesticidal proteins are sufficiently identical to the aminoacid sequence set forth in SEQ ID NO:2. By “sufficiently identical” isintended an amino acid or nucleotide sequence that has at least about60% or 65% sequence identity, about 70% or 75% sequence identity, about80% or 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or greater sequence identity compared to a referencesequence using one of the alignment programs described herein usingstandard parameters. One of skill in the art will recognize that thesevalues can be appropriately adjusted to determine corresponding identityof proteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the percent identity iscalculated across the entirety of the reference sequence. The percentidentity between two sequences can be determined using techniquessimilar to those described below, with or without allowing gaps. Incalculating percent identity, typically exact matches are counted. Agap, (a position in an alignment where a residue is present in onesequence but not in the other) is regarded as a position withnon-identical residues.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Nat'l. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Nat'l. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, word length=12, to obtain nucleotide sequences homologous topesticidal-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4(1):11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., San Diego, Calif., USA). When utilizing the ALIGNprogram for comparing amino acid sequences, a PAM120 weight residuetable, a gap length penalty of 12, and a gap penalty of 4 can be used.Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

Protease sensitivity variants. VIP3 proteins, including DIG-657, may beproteolytically truncated from about 88 kDa in size to a product ofabout 66 kDa in size. The 66 kDa protein comprises amino acid residues206-803. Insect gut proteases typically function in aiding the insect inobtaining needed amino acids from dietary protein. The best understoodinsect digestive proteases are serine proteases, which appear to be themost common type (Englemann and Geraerts (1980), particularly inLepidopteran species. Coleopteran insects have guts that are moreneutral to acidic than are Lepidopteran guts. The majority ofColeopteran larvae and adults, for example Colorado potato beetle (CPB),have slightly acidic midguts, and cysteine proteases provide the majorproteolytic activity (Wolfson and Murdock, 1990). More precisely, Thieand Houseman (1990) identified and characterized the cysteine proteases,cathepsin B-like and cathepsin H-like, and the aspartyl protease,cathepsin D-like, in CPB. Gillikin et al. (1992) characterized theproteolytic activity in the guts of WCR larvae and found primarilycysteine proteases. U.S. Pat. No. 7,230,167 disclosed that a proteaseactivity attributed to cathepsin G exists in WCR. The diversity anddifferent activity levels of the insect gut proteases may influence aninsect's sensitivity to a particular Bt toxin.

In another embodiment of the invention, protease cleavage sites may beengineered at desired locations to affect protein processing within themidgut of susceptible larvae of certain insect pests. These proteasecleavage sites may be introduced by methods such as chemical genesynthesis or splice overlap PCR (Horton et al., 1989). Serine proteaserecognition sequences, for example, can optionally be inserted atspecific sites in the Cry protein structure to affect protein processingat desired deletion points within the midgut of susceptible larvae.Serine proteases that can be exploited in such fashion includeLepidopteran midgut serine proteases such as trypsin or trypsin-likeenzymes, chymotrypsin, elastase, etc. (Christeller et al., 1992).Further, deletion sites identified empirically by sequencing Cry proteindigestion products generated with unfractionated larval midgut proteasepreparations or by binding to brush border membrane vesicles can beengineered to effect protein activation. Modified Cry or VIP3 proteinsgenerated either by gene deletion or by introduction of proteasecleavage sites having improved activity on Lepidopteran pests includingECB, CEW, CBW, BCW, FAW, BAW, Diatraea grandiosella, Diatraeasaccharalis, Loxagrotis albicosta, and other target pests.

Lepidopteran and Coleopteran serine proteases such as trypsin,chymotrypsin and cathepsin G-like protease, Lepidopteran and Coleopterancysteine proteases such as cathepsins (B-like, L-like, O-like, andK-like proteases) (Koiwa et al., (2000) and Bown et al., (2004)),Lepidopteran and Coleopteran metalloproteases such as ADAM10(Ochoa-Campuzano et al., (2007)), and Lepidoperan and Coleopteranaspartic acid proteases such as cathepsins D-like and E-like, pepsin,plasmepsin, and chymosin may further be exploited by engineeringappropriate recognition sequences at desired processing sites to affectCry protein processing within the midgut of susceptible larvae ofcertain insect pests and perhaps also function to provide activityagainst non-susceptible insect pests.

DIG-657 variants produced by introduction or elimination of proteaseprocessing sites at appropriate positions in the coding sequence toallow, or eliminate, proteolytic cleavage of a larger variant protein byinsect, plant, or microorganism proteases are within the scope of theinvention. The end result of such manipulation is understood to be thegeneration of toxin fragment molecules having the same or betteractivity as the intact (full length) toxin protein.

Spray-on applications are another example and are also known in the art.The subject proteins can be appropriately formulated for the desired enduse, and then sprayed (or otherwise applied) onto the plant and/oraround the plant and/or to the vicinity of the plant to beprotected—before an infestation is discovered, after target insects arediscovered, both before and after, and the like. Bait granules, forexample, can also be used and are known in the art.

When an insect comes into contact with an effective amount of toxindelivered via transgenic plant expression, formulated proteincomposition(s), sprayable protein composition(s), a bait matrix or otherdelivery system, the results are typically death of the insect, or theinsects do not feed upon the source which makes the toxins available tothe insects.

With suitable microbial hosts, e.g. Pseudomonas, the microbes can beapplied to the environment of the pest, where they will proliferate andbe ingested. The result is control of the pest. Alternatively, themicrobe hosting the toxin gene can be treated under conditions thatprolong the activity of the toxin and stabilize the cell. The treatedcell, which retains the toxic activity, then can be applied to theenvironment of the target pest.

Development of Oligonucleotide Probes. A further method for identifyingthe toxins and genes of the subject invention is through the use ofoligonucleotide probes. These probes are detectable nucleotidesequences. These sequences may be rendered detectable by virtue of anappropriate radioactive label or may be made inherently fluorescent asdescribed in, for example, U.S. Pat. No. 6,268,132. As is well known inthe art, if the probe molecule and nucleic acid sample hybridize byforming strong base-pairing bonds between the two molecules, it can bereasonably assumed that the probe and sample have substantial sequencehomology. Preferably, hybridization is conducted under stringentconditions by techniques well-known in the art, as described, forexample, in Keller and Manak (1993). Detection of the probe provides ameans for determining in a known manner whether hybridization hasoccurred. Such a probe analysis provides a rapid method for identifyingtoxin-encoding genes of the subject invention. The nucleotide segmentswhich are used as probes according to the invention can be synthesizedusing a DNA synthesizer and standard procedures. These nucleotidesequences can also be used as PCR primers to amplify genes of thesubject invention.

Nucleic acid hybridization. As is well known to those skilled inmolecular biology, similarity of two nucleic acids can be characterizedby their tendency to hybridize. As used herein the terms “stringentconditions” or “stringent hybridization conditions” are intended torefer to conditions under which a probe will hybridize (anneal) to itstarget sequence to a detectably greater degree than to other sequences(e.g. at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to pH 8.3 and thetemperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C. and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate)at 50° C. to 55° C. Exemplary moderate stringency conditions includehybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C. anda wash in 0.5× to 1×SSC at 55° C. to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C. and a wash in 0.1×SSC at 60° C. to 65° C. Optionally, washbuffers may comprise about 0.1% to about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 to12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA/DNA hybrids, the thermal melting point (T_(m)) isthe temperature (under defined ionic strength and pH) at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization conditions, and/or wash conditions can be adjusted tofacilitate annealing of sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, highly stringent conditions canutilize a hybridization and/or wash at 1° C., 2° C., 3° C., or 4° C.lower than the T_(m); moderately stringent conditions can utilize ahybridization and/or wash at 6° C., 7° C., 8° C., 9° C., or 10° C. lowerthan the T_(m), and low stringency conditions can utilize ahybridization and/or wash at 11° C., 12° C., 13° C., 14° C., 15° C., or20° C. lower than the T_(m).

T_(m) (in ° C.) may be experimentally determined or may be approximatedby calculation. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984): T_(m)(° C.)=81.5° C.+16.6(logM)+0.41(% GC)−0.61(% formamide)−500/L; where M is the molarity ofmonovalent cations, % GC is the percentage of guanosine and cytosinenucleotides in the DNA, % formamide is the percentage of formamide inthe hybridization solution, and L is the length of the hybrid in basepairs. Alternatively, the T_(m) is described by the following formula(Beltz et al., 1983): T_(m)(° C.)=81.5° C.+16.6(log[Na+])+0.41(%GC)−0.61(% formamide)−600/L where [Na+] is the molarity of sodium ions,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% formamide is the percentage of formamide in the hybridizationsolution, and L is the length of the hybrid in base pairs.

Using the equations, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Hybridization with Nucleic Acid Probes Vol. 1by P. Tijssen (1993, ISBN-10: 0444898840, ISBN-13: 9780444898845Hardcover) and Ausubel et al. (1995). Also see Sambrook et al. (1989).

EXAMPLES Example 1

Construction of expression plasmids encoding DIG-657 insecticidal toxinand expression in bacterial hosts. Standard cloning methods were used inthe construction of Pseudomonas fluorescens (Pf) expression plasmidsengineered to produce full-length DIG-657 proteins encoded byplant-optimized coding regions. Restriction endonucleases and T4 DNALigase were obtained from New England BioLabs (NEB; Ipswich, Mass.) forrestriction digestion and DNA ligation, respectively. Plasmidpreparations were performed using the NucleoSpin® Plasmid Kit(Macherey-Nagel Inc, Bethlehem, Pa.) following the instructions of thesuppliers for low-copy plasmid purification. DNA fragments were purifiedusing a QIAquick® Gel Extraction Kit (Qiagen, Venio, Limburg) afteragarose Tris-acetate gel electrophoresis.

The gene encoding DIG-657 was amplified out of the parental Bt StrainPS46L/DBt11889 using gene specific primers containing specificrestriction sites for cloning. The resulting DIG-657 Polymerase ChainReaction (PCR) product was then cloned into Pseudomonas fluorescensexpression vector pDOW1169 using the traditional ligation cloningmethod.

The basic cloning strategy entailed subcloning the DIG-657 toxin codingsequence (CDS) (SEQ ID NO:1) into pDOW1169 at the Spe1 and Sal1restriction sites, whereby it is placed under the expression control ofthe Ptac promoter and the rrnBT1T2 terminator from plasmid pKK223-3 (PLPharmacia, Milwaukee, Wis.). pDOW1169 is a medium copy plasmid with theRSF1010 origin of replication, a pyrF gene, and a ribosome binding sitepreceding the restriction enzyme recognition sites into which DNAfragments containing protein coding regions may be introduced, (USApplication 20080193974). The expression plasmid, designated pDOW1169,was transformed by electroporation into DC454 (a near wild-type P.fluorescens strain having mutations deltapyrF and lsc::lacI^(QI)), orits derivatives, recovered in SOC-Soy hydrolysate medium, and plated onselective medium (M9 glucose agar lacking uracil, Sambrook et al.,supra). Details of the microbiological manipulations are available inSquires et al., (2004), US Patent Application 20060008877, US PatentApplication 20080193974, and US Patent Application 20080058262,incorporated herein by reference. Colonies were first screened byrestriction digestion of miniprep plasmid DNA. Plasmid DNA of selectedclones containing DIG-657 toxin were digested with four restrictionenzymes and sequence verified to further validate presence of theinsert.

Example 2

Growth and Expression Analysis in Shake Flasks. Production of DIG-657toxin for characterization and insect bioassay was accomplished byshake-flask-grown P. fluorescens strains harboring expression constructs(pDOW1169). A glycerol stock of DIG-657 culture (0.5 mL) was inoculatedinto 50 mL of defined production medium with 9.5% glycerol (TeknovaCatalog No. 3D7426, Hollister, Calif.). Expression of the DIG-657 toxingene via the Ptac promoter was induced by addition ofisopropyl-β-D-1-thiogalactopyranoside (IPTG) after an initial incubationof 24 hours at 30° C. with shaking. Cultures were sampled at the time ofinduction and at various times post-induction. Cell density was measuredby optical density at 600 nm (OD₆₀₀). Other culture media suitable forgrowth of Pseudomonas fluorescens may also be utilized, for example, asdescribed in Huang et al. (2007) and US Patent Application 20060008877.

Example 3

Agrobacterium transformation. Standard cloning methods were used in theconstruction of binary plant transformation and expression plasmids.Restriction endonucleases and T4 DNA Ligase were obtained from NEB.Plasmid preparations were performed using the NucleoSpin® PlasmidPreparation kit or the NucleoBond® AX Xtra Midi kit (both fromMacherey-Nagel, Duren, Germany), following the instructions of themanufacturers. DNA fragments were purified using the QIAquick® PCRPurification Kit or the QIAEX II® Gel Extraction Kit (both from QiagenVenio, Limburg) after gel isolation.

Electro-competent cells of Agrobacterium tumefaciens strain Z7075 (astreptomycin-resistant derivative of Z707; Hepburn et al., 1985) wereprepared and transformed using electroporation (Weigel and Glazebrook,2002). After electroporation, 1 mL of Yeast Extract Peptone (YEP) broth(10 gm/L yeast extract, 10 gm/L peptone, and 5 gm/L NaCl) was added tothe cuvette and the cell-YEP suspension was transferred to a 15 mLculture tube for incubation at 28° C. in a water bath with constantagitation for 4 hours. The cells were plated on YEP plus agar (25 gm/L)with spectinomycin (200 μg/mL) and streptomycin (250 μg/mL) and theplates were incubated for 2-4 days at 28° C. Well separated singlecolonies were selected and streaked onto fresh YEP+agar plates withspectinomycin and streptomycin as described above, and incubated at 28°C. for 1-3 days.

The presence of the DIG-657 gene insert in the binary planttransformation vector was performed by PCR analysis usingvector-specific primers with template plasmid DNA prepared from selectedAgrobacterium colonies. The cell pellet from a 4 mL aliquot of a 15 mLovernight culture grown in YEP with spectinomycin and streptomycin asbefore was extracted using Qiagen (Venlo, Limburg, Netherlands) Spin®Mini Preps, performed per manufacturer's instructions. Plasmid DNA fromthe binary vector used in the Agrobacterium electroporationtransformation was included as a control. The PCR reaction was completedusing Taq DNA polymerase from Invitrogen (Carlsbad, Calif.) permanufacturer's instructions at 0.5× concentrations. PCR reactions werecarried out in a MJ Research Peltier Thermal Cycler programmed with thefollowing conditions: Step 1) 94° C. for 3 minutes; Step 2) 94° C. for45 seconds; Step 3) 55° C. for 30 seconds; Step 4) 72° C. for 1 minuteper kb of expected product length; Step 5) 29 times to Step 2; Step 6)72° C. for 10 minutes. The reaction was maintained at 4° C. aftercycling. The amplification products were analyzed by agarose gelelectrophoresis (e.g. 0.7% to 1% agarose, w/v) and visualized byethidium bromide staining. A colony was selected whose PCR product wasidentical to the plasmid control.

Example 4

Production of DIG-657. Cells of shake-flask-grown P. fluorescens strainsharboring expression constructs (pDOW1169) were isolated bycentrifugation and the resulting cell pellets frozen at −80° C. Solubleand insoluble fractions from frozen shake flask cell pellet samples weregenerated using EasyLyse™ Bacterial Protein Extraction Solution(EPICENTRE® Biotechnologies, Madison, Wis.). Each cell pellet wassuspended into 1 mL EasyLyse™ solution and further diluted 1:4 in lysisbuffer and incubated with shaking at room temperature for 30 minutes.The lysate was centrifuged at 14,000 rpm for 20 minutes at 4° C. and thesupernatant was recovered as the soluble fraction. The pellet (insolublefraction) was then suspended in an equal volume of phosphate bufferedsaline (PBS; 11.9 mM Na₂HPO₄, 137 mM NaCl, 2.7 mM KCl, pH7.4).

Samples were mixed 1:1 with 2× Laemmli sample buffer containingβ-mercaptoethanol (Sambrook et al., supra.) and boiled for 5 minutesprior to loading onto Criterion XT® Bis-Tris 12% gels (Bio-Rad Inc.,Hercules, Calif.). Electrophoresis was performed in the recommended XTMOPS buffer. Gels were stained with Bio-Safe Coomassie Stain (Bio-Rad,Richmond, Calif.) according to the manufacturer's protocol and imagedusing the Alpha Innotech Imaging system (San Leandro, Calif.).

Example 5

Inclusion body preparation. DIG-657 was generally located in the solublefraction of P. fluorescens cells containing the gene for DIG-657. Insome cases, a variable percentage of the DIG-657 protein was foundlocated in protein inclusion body (IB) preparations, as demonstrated bySDS-PAGE and MALDI-MS (Matrix Assisted Laser Desorption/Ionization MassSpectrometry). To isolate this protein from this fraction, P.fluorescens fermentation pellets were thawed in a 37° C. water bath. Thecells were resuspended to 25% w/v in lysis buffer (50 mM Tris, pH 7.5,200 mM NaCl, 20 mM EDTA disodium salt (Ethylenediaminetetraacetic acid),1% Triton X-100, and 5 mM Dithiothreitol (DTT)); 5 mL/L of bacterialprotease inhibitor cocktail (P8465 Sigma-Aldrich, St. Louis, Mo.) wereadded just prior to use. The cells were suspended using a hand-heldhomogenizer at the lowest setting (Tissue Tearor, BioSpec Products,Inc., Bartlesville, Okla.). Lysozyme (25 mg of Sigma, St. Louis, Mo.,L7651 from chicken egg white) was added to the cell suspension by mixingwith a metal spatula, and the suspension was incubated at roomtemperature for one hour. The suspension was cooled on ice for 15minutes, then sonicated using a Branson (Danbury, Conn.) Sonifier 250(two 1-minute sessions, at 50% duty cycle, 30% output). Cell lysis waschecked by microscopy. An additional 25 mg of lysozyme were added ifnecessary, and the incubation and sonication steps were repeated. Whencell lysis was confirmed via microscopy, the lysate was centrifuged at11,500×g for 25 minutes (4° C.) to form the IB pellet, and thesupernatant was discarded. The IB pellet was resuspended with 100 mLlysis buffer, homogenized with the hand-held mixer and centrifuged asabove. The IB pellet was repeatedly washed by resuspension (in 50 mLlysis buffer), homogenization, sonication, and centrifugation until thesupernatant became colorless and the IB pellet became firm and off-whitein color. For the final wash, the IB pellet was resuspended insterile-filtered (0.22 μm) distilled water containing 2 mM EDTA, andcentrifuged. The final pellet was resuspended in sterile-filtereddistilled water containing 2 mM EDTA, and stored in 1 mL aliquots at−80° C.

SDS-PAGE analysis and quantification of protein in IB preparations wasdone by thawing a 1 mL aliquot of IB pellet and diluting 1:20 withsterile-filtered distilled water. The diluted sample was then boiledwith 4× reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v),0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% β-Mercapto-ethanol(v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel(Invitrogen, Carlsbad, Calif.) run with 1×Tris/Glycine/SDS buffer(BioRad, Richmond, Calif.). The gel was run for 60 min at 200 volts thenstained with Coomassie Blue (50% G-250/50% R-250 in 45% methanol, 10%acetic acid), and destained with 7% acetic acid, 5% methanol indistilled water. Quantification of target bands was done by comparingdensitometric values for the bands against Bovine Serum Albumin (BSA)samples run on the same gel to generate a standard curve.

Example 6

Solubilization of Inclusion Bodies. Six mL of inclusion body suspension(containing 32 mg/mL of DIG-657 protein) were centrifuged on the highestsetting of an Eppendorf model 5415C microfuge (approximately 14,000×g)to pellet the inclusions. The storage buffer supernatant was removed andreplaced with 25 mL of 100 mM sodium carbonate buffer, pH11, in a 50 mLconical tube. Inclusions were resuspended using a pipette and vortexedto mix thoroughly. The tube was placed on a gently rocking platform at4° C. overnight to extract the target protein. The extract wascentrifuged at 30,000×g for 30 min at 4° C., and the resultingsupernatant was concentrated 5-fold using an Amicon Ultra-15 regeneratedcellulose centrifugal filter device (30,000 Molecular Weight Cutoff;Millipore, Billerica, Mass.). The sample buffer was then changed to 10mM CAPS [3-(cyclohexamino)1-propanesulfonic acid] pH 10, usingdisposable PD-10 columns (GE Healthcare, Piscataway, N.J.).

Example 7

Gel electrophoresis. The concentrated extract was prepared forelectrophoresis by diluting 1:50 in NuPAGE® LDS sample buffer(Invitrogen, Carlsbad, Calif.) containing κ mM dithiothreitol as areducing agent and heated at 95° C. for 4 minutes. The sample was loadedin duplicate lanes of a 4-12% NuPAGE® gel alongside five BSA standardsranging from 0.2 to 2 μg/lane (for standard curve generation). Voltagewas applied at 200V using MOPS SDS running buffer (Invitrogen, Carlsbad,Calif.) until the tracking dye reached the bottom of the gel. The gelwas stained with 0.2% Coomassie Blue G-250 in 45% methanol, 10% aceticacid, and destained, first briefly with 45% methanol, 10% acetic acid,and then at length with 7% acetic acid, 5% methanol until the backgroundcleared. Following destaining, the gel was scanned with a Biorad Fluor-SMultilmager. The instrument's Quantity One v.4.5.2 Software was used toobtain background-subtracted volumes of the stained protein bands and togenerate the BSA standard curve that was used to calculate theconcentration of DIG-657 protein in the stock solution.

Example 8

DIG-657 purification. Purification of DIG-657 was conducted similar toVIP3 as described by Lee, M. K., et al (2003), where cells from Pftransformed with the DIG-657 gene were defrosted, suspended intoextraction buffer (10 mM Tris-HCl, 2 mM EDTA, 2 mM DTT) in the presenceof 0.5 ml protease inhibitor cocktail (Sigma, St. Louis, Mo.) andsonicated five times for one min. each, with resting on ice for 1 min.between each sonication cycle. The solution was centrifuged at 20,000×gfor 10 min., and the supernatant collected and applied to a MonoQ ionexchange column (1 cm dia.×10 cm long). Protein purification wasachieved by sequential chromatography on ion exchange (MonoQ 1010, GEHealthcare, Piscataway, N.J.), hydrophobic chromatography, and sizeexclusion chromatography. The final preparation showed a single band ofprotein migrating at an apparent molecular weight of about 90 kDa.

Example 9

Sample preparation and bioassays. Purified preparations of DIG-657 werediluted appropriately in 10 mM CAPS, pH 10, and all bioassays containeda control treatment consisting of this buffer, which served as abackground check for mortality and/or growth inhibition. Proteinconcentrations in bioassay buffer were estimated by gel electrophoresisusing bovine serum albumin (BSA) to create a standard curve for geldensitometry, which was measured using a BioRad (Richmond, Calif.)imaging system (Fluor-S Multilmager with Quantity One software version4.5.2). Proteins in the gel matrix were stained with CoomassieBlue-based stain and destained before reading.

Purified proteins were tested for insecticidal activity in bioassaysconducted with 1-2 day old neonate Lepidopteran larvae on artificialinsect diet. Larvae of CEW, SBL, DBM, VBC, ECB, FAW and TBW were hatchedfrom eggs obtained from a colony maintained by a commercial insectary(Benzon Research Inc., Carlisle, Pa.). Larvae of rECB and rFAW werehatched from eggs harvested from proprietary colonies (Dow AgroSciencesLLC, Indianapolis, Ind.).

The bioassays were conducted in 128-well plastic trays specificallydesigned for insect bioassays (C-D International, Pitman, N.J.). Eachwell contained 2.0 mL of Multi-species Lepidoptera diet (SouthlandProducts, Lake Village, Ark.). A 40 μL aliquot of protein sample wasdelivered by pipette onto the 2.0 cm² diet surface of each well (20μL/cm²). Diet concentrations were calculated as the amount (ng) ofDIG-657 protein per square centimeter (cm²) of surface area in the well.A 9 dose concentration range was used from 9,000 to 3 ng/cm² with 16larvae tested per dose. The treated trays were held in a fume hood untilthe liquid on the diet surface had evaporated or was absorbed into thediet.

Within a 24-48 hours of eclosion, individual larvae were picked up witha moistened camel hair brush and deposited on the treated diet, onelarva per well. The infested wells were then sealed with adhesive sheetsof clear plastic and vented to allow gas exchange (C-D International,Pitman, N.J.). Bioassay trays were held under controlled environmentalconditions (28° C., ˜60% Relative Humidity, 16:8 [Light:Dark]) for 5days, after which the total number of insects exposed to each proteinsample, the number of dead insects, and the weight of surviving insectswere recorded. Percent mortality and percent growth inhibition werecalculated for each treatment. Growth inhibition (GI) was calculatedusing the formula below:GI=[1−(TWIT/TNIT)/(TWIBC/TNIBC)]where TWIT is the Total Weight of Insects in the Treatment, TNIT is theTotal Number of Insects in the Treatment, TWIBC is the Total Weight ofInsects in the Background Check (Buffer control), and TNIBC is the TotalNumber of Insects in the Background Check (Buffer control).

The GI₅₀ was determined to be the concentration of DIG-657 protein inthe diet at which the GI value was 50%. The 50% lethal concentration(LC50) was recorded as the concentration of DIG-657 protein in the dietat which 50% of test insects were killed. Growth inhibitionconcentration-response curves were determined by using a nonlinearlogistic 3-parameter through JMP Pro, version 9.0.3, software (SASInstitute Inc., Cary, N.C.). Lethal concentration-response curve wereanalyzed by Probit analyses (Finney, 1971) of the pooled mortality dataand were conducted using POLO-PC (LeOra Software).

Table 2 presents the results of bioassay tests of DIG-657 protein onECB, rECB, TBW, Pseudoplusia includens (Walker) (soybean looper, “SBL”),Anticarsia gemmatalis (Hubner) (velvetbean caterpillar, “VBC”), CEW,FAW, rFAW, and DBM. An unexpected and surprising finding is that therECB test insects were as susceptible if not more to the action ofDIG-657 protein as were the susceptible strain of ECB insects.

TABLE 2 Efficacy of purified DIG-657 against multiple insect speciestested on artificial diet-overlay bioassay (ng/cm²). Insect LC₅₀ (CI95%)(ng/cm²) GI₅₀ (CI 95%)(ng/cm²) ECB 281 (166-470) 8.6 (6.6-11.1) rECB37 (17-74) 8.1 (4.2-15.9) TBW 608 (311-1,432) 12.9 (5.9-28.1) SBL 80(50-120) 5.4 (4.3-6.7) VBC 66 (45-95) 18.9 (12.2-29.3) CEW >9,000 17.5(10.9-28.2) FAW >9,000 563 (398-796) rFAW ~9,000 2,651 (1,030-6,822) DBM6.3 (1.6-11.8) 3.7 (3.0-4.6)

Helicoverpa armigera bioassays were conducted by surface contamination,using neonate larvae. Toxicity tests were performed in 128-cell trays,each cell 2 cm². Concentrations of 25 and 2500 ng/cm² were used todetermine percent mortality, with a control of buffer only (Table3).Tests to determine LC₅₀ values were performed using 7 differentconcentrations of purified protoxins, with a control of only buffer(Table 4). Mortality and arrest were assessed after 7 days at 25° C.with 16:8 light:dark conditions. “Functional mortality” was obtainedscoring dead and L1 arrested larvae. Results showed DIG-657 activityagainst Helicoverpa armigera.

TABLE 3 Percent mortality of DIG-657 against Helicoverpa armigera testedon artificial diet bioassay. Mortality % Functional Mortality % 25ng/cm² 2500 ng/cm² 25 ng/cm² 2500 ng/cm² Cry1Ac 28 92 33 100 Cry1Fa 6 216 21 DIG-657 38 79 38 93

TABLE 4 LC₅₀ (ng/cm²) of DIG-657 against Helicoverpa armigera tested onartificial diet bioassay. Mortality Functional Mortality Toxin RepsLC₅₀* Slope LC₅₀* Slope Cry1Ac 3 13.6 (0.3-44.9) 0.65 ± 0.12 5.05(0.02-19.27) 0.87 ± 0.16 DIG-657 5 3389 (963-79246) 0.56 ± 0.08 301(183-541) 0.84 ± 0.08 *Values in ( ) are 0.95 confidence limits

Example 10 Production of DIG-657 Bt Insecticidal Proteins and Variantsin Dicot Plants

Arabidopsis Transformation.

Arabidopsis thaliana Col-01 is transformed using the floral dip method(Weigel and Glazebrook, 2002). The selected Agrobacterium colony is usedto inoculate 1 mL to 15 mL cultures of YEP broth containing appropriateantibiotics for selection. The culture is incubated overnight at 28° C.with constant agitation at 220 rpm. Each culture is used to inoculatetwo 500 mL cultures of YEP broth containing appropriate antibiotics forselection and the new cultures are incubated overnight at 28° C. withconstant agitation. The cells are pelleted at approximately 8700×g for10 minutes at room temperature, and the resulting supernatant isdiscarded. The cell pellet is gently resuspended in 500 mL ofinfiltration media containing: ½× Murashige and Skoog salts(Sigma-Aldrich)/Gamborg's B5 vitamins (Gold BioTechnology, St. Louis,Mo.), 10% (w/v) sucrose, 0.044 μM benzylaminopurine (10 μL/L of 1 mg/mLstock in DMSO) and 300 μL/L Silwet L-77. Plants approximately 1 monthold are dipped into the media for 15 seconds, with care taken to assuresubmergence of the newest inflorescence. The plants are then laid ontheir sides and covered (transparent or opaque) for 24 hours, washedwith water, and placed upright. The plants are grown at 22° C., with a16:8 light:dark photoperiod. Approximately 4 weeks after dipping, theseeds are harvested.

Arabidopsis Growth and Selection.

Freshly harvested T1 seed is allowed to dry for at least 7 days at roomtemperature in the presence of desiccant. Seed is suspended in a 0.1%agar/water (Sigma-Aldrich) solution and then stratified at 4° C. for 2days. To prepare for planting, Sunshine Mix LP5 (Sun Gro HorticultureInc., Bellevue, Wash.) in 10.5 inch×21 inch germination trays (T.O.Plastics Inc., Clearwater, Minn.) is covered with fine vermiculite,sub-irrigated with Hoagland's solution (Hoagland and Arnon, 1950) untilwet, then allowed to drain for 24 hours. Stratified seed is sown ontothe vermiculite and covered with humidity domes (KORD Products,Bramalea, Ontario, Canada) for 7 days. Seeds are germinated and plantsare grown in a Conviron (Models CMP4030 or CMP3244; ControlledEnvironments Limited, Winnipeg, Manitoba, Canada) under long dayconditions (16:8 light:dark photoperiod) at a light intensity of 120-150mol/m²sec under constant temperature (22° C.) and humidity (40-50%).Plants are initially watered with Hoagland's solution and subsequentlywith deionized water to keep the soil moist but not wet.

The domes are removed 5-6 days post sowing and plants are sprayed with achemical selection agent to kill plants germinated from nontransformedseeds. For example, if the plant expressible selectable marker geneprovided by the binary plant transformation vector is a pat or bar gene(Wehrmann et al., 1996), transformed plants may be selected by sprayingwith a 1000× solution of Finale (5.78% glufosinate ammonium, FarnamCompanies Inc., Phoenix, Ariz.). Two subsequent sprays are performed at5-7 day intervals. Survivors (plants actively growing) are identified7-10 days after the final spraying and are transplanted into potsprepared with Sunshine Mix LP5. Transplanted plants are covered with ahumidity dome for 3-4 days and placed in a Conviron under theabove-mentioned growth conditions.

Those skilled in the art of dicot plant transformation will understandthat other methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g. herbicidetolerance genes) are used.

Example 11

Transgenic Glycine max Comprising DIG-657.

Ten to 20 transgenic T₀ Glycine max plants harboring expression vectorsfor nucleic acids comprising DIG-657 are generated as is known in theart, including for example by Agrobacterium-mediated transformation.Mature soybean (Glycine max) seeds are sterilized overnight withchlorine gas for sixteen hours. Following sterilization with chlorinegas, the seeds are placed in an open container in a LAMINAR™ flow hoodto dispel the chlorine gas. Next, the sterilized seeds are imbibed withsterile H₂O for sixteen hours in the dark using a black box at 24° C.

Preparation of split-seed soybeans. The split soybean seed comprising aportion of an embryonic axis protocol requires preparation of soybeanseed material which is cut longitudinally, using a #10 blade affixed toa scalpel, along the hilum of the seed to separate and remove the seedcoat, and to split the seed into two cotyledon sections. Carefulattention is made to partially remove the embryonic axis, wherein about½-⅓ of the embryo axis remains attached to the nodal end of thecotyledon.

Inoculation. The split soybean seeds comprising a partial portion of theembryonic axis are then immersed for about 30 minutes in a solution ofAgrobacterium tumefaciens (e.g., strain EHA 101 or EHA 105) containingbinary plasmid comprising DIG-657. The Agrobacterium tumefacienssolution is diluted to a final concentration of λ=0.6 OD₆₅₀ beforeimmersing the cotyledons comprising the embryo axis.

Co-cultivation. Following inoculation, the split soybean seed is allowedto co-cultivate with the Agrobacterium tumefaciens strain for 5 days onco-cultivation medium (Wang, Kan. Agrobacterium Protocols. 2. 1. NewJersey: Humana Press, 2006. Print.) in a Petri dish covered with a pieceof filter paper.

Shoot induction. After 5 days of co-cultivation, the split soybean seedsare washed in liquid Shoot Induction (SI) media consisting of B5 salts,B5 vitamins, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 100 mg/L TIMENTIN™, 200 mg/L cefotaxime, and 50 mg/Lvancomycin (pH 5.7). The split soybean seeds are then cultured on ShootInduction I (SI I) medium consisting of B5 salts, B5 vitamins, 7 g/LNoble agar, 28 mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose, 0.6 g/LMES, 1.11 mg/L BAP, 50 mg/L TIMENTIN™, 200 mg/L cefotaxime, 50 mg/Lvancomycin (pH 5.7), with the flat side of the cotyledon facing up andthe nodal end of the cotyledon imbedded into the medium. After 2 weeksof culture, the explants from the transformed split soybean seed aretransferred to the Shoot Induction II (SI II) medium containing SI Imedium supplemented with 6 mg/L glufosinate (LIBERTY®).

Shoot elongation. After 2 weeks of culture on SI II medium, thecotyledons are removed from the explants and a flush shoot padcontaining the embryonic axis are excised by making a cut at the base ofthe cotyledon. The isolated shoot pad from the cotyledon is transferredto Shoot Elongation (SE) medium. The SE medium consists of MS salts, 28mg/L Ferrous, 38 mg/L Na₂EDTA, 30 g/L sucrose and 0.6 g/L MES, 50 mg/Lasparagine, 100 mg/L L-pyroglutamic acid, 0.1 mg/L IAA, 0.5 mg/L GA3, 1mg/L zeatin riboside, 50 mg/L TIMENTIN™, 200 mg/L cefotaxime, 50 mg/Lvancomycin, 6 mg/L glufosinate, 7 g/L Noble agar, (pH 5.7). The culturesare transferred to fresh SE medium every 2 weeks. The cultures are grownin a CONVIRON™ growth chamber at 24° C. with an 18 h photoperiod at alight intensity of 80-90 mol/m²sec.

Rooting. Elongated shoots which developed from the cotyledon shoot padare isolated by cutting the elongated shoot at the base of the cotyledonshoot pad, and dipping the elongated shoot in 1 mg/L IBA (Indole3-butyric acid) for 1-3 minutes to promote rooting. Next, the elongatedshoots are transferred to rooting medium (MS salts, B5 vitamins, 28 mg/LFerrous, 38 mg/L Na₂EDTA, 20 g/L sucrose and 0.59 g/L MES, 50 mg/Lasparagine, 100 mg/L L-pyroglutamic acid 7 g/L Noble agar, pH 5.6) inphyta trays.

Cultivation. Following culture in a CONVIRON™ growth chamber at 24° C.,18 h photoperiod, for 1-2 weeks, the shoots which have developed rootsare transferred to a soil mix in a covered sundae cup and placed in aCONVIRON™ growth chamber (models CMP4030 and CMP3244, ControlledEnvironments Limited, Winnipeg, Manitoba, Canada) under long dayconditions (16 hours light/8 hours dark) at a light intensity of 120-150μmol/m²sec under constant temperature (22° C.) and humidity (40-50%) foracclimatization of plantlets. The rooted plantlets are acclimated insundae cups for several weeks before they are transferred to thegreenhouse for further acclimatization and establishment of robusttransgenic soybean plants.

Development and morphological characteristics of transgenic lines arecompared with nontransformed plants. Plant root, shoot, foliage andreproduction characteristics are compared. There are no observabledifference in root length and growth patterns of transgenic andnontransformed plants. Plant shoot characteristics such as height, leafnumbers and sizes, time of flowering, floral size and appearance aresimilar. In general, there are no observable morphological differencesbetween transgenic lines and those without expression of DIG proteinswhen cultured in vitro and in soil in the glasshouse.

Example 12 Production of DIG-657 Bt Insecticidal Proteins and Variantsin Monocot Plants

Agrobacterium-Mediated Transformation of Maize.

Seeds from a High II or B-104 F₁ cross (Armstrong et al., 1991) wereplanted into 5-gallon-pots containing a mixture of 95% Metro-Mix 360soilless growing medium (Sun Gro Horticulture, Bellevue, Wash.) and 5%clay/loam soil. The plants were grown in a greenhouse using acombination of high pressure sodium and metal halide lamps with a 16:8hour light:dark photoperiod. To obtain immature F2 embryos fortransformation, controlled sib-pollinations were performed. Immatureembryos were isolated at 8-10 days post-pollination when embryos wereapproximately 1.0 to 2.0 mm in size.

Infection and co-cultivation Maize ears were surface sterilized byscrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, andthen immersing in 20% commercial bleach (0.1% sodium hypochlorite) for30 minutes before being rinsed with sterile water.

A suspension of Agrobacterium cells containing a superbinary vector wereprepared by transferring 1-2 loops of bacteria grown on YEP solid mediumcontaining 100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/Lstreptomycin at 28° C. for 2-3 days into 5 mL of liquid infection medium(LS Basal Medium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al.,1975), 1.5 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/Lsucrose, 36.0 gm/L glucose, 6 mM L-proline, pH 5.2) containing 100 μMacetosyringone. The solution was vortexed until a uniform suspension wasachieved, and the concentration was adjusted to a final density of about200 Klett units, using a Klett-Summerson colorimeter with a purplefilter, or an optical density of approximately 0.4 at 550 nm. Immatureembryos were isolated directly into a micro centrifuge tube containing 2mL of the infection medium. The medium was removed and replaced with 1mL of the Agrobacterium solution with a density of 200 Klett units, andthe Agrobacterium and embryo solution was incubated for 5 minutes atroom temperature and then transferred to co-cultivation medium (LS BasalMedium, (containing N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mML-proline, 0.85 mg/L AgNO₃, 100 μM acetosyringone, and 3.0 gm/L Gellangum (PhytoTechnology Laboratories, Lenexa, Kans.), pH 5.8) for 5 days at25° C. under dark conditions.

After co-cultivation, the embryos were transferred to selective mediumafter which transformed isolates were obtained over the course ofapproximately 8 weeks. For selection of maize tissues transformed with asuperbinary plasmid containing a plant expressible pat or bar selectablemarker gene, an LS based medium (LS Basal medium, with 1×N6 vitamins,1.5 mg/L 2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acidmonohydrate; PhytoTechnology Laboratories., Lenexa, Kans.), 30.0 gm/Lsucrose, 6 mM L-proline, 1.0 mg/L AgNO₃, 250 mg/L cefotaxime, 2.5 gm/LGellan gum, pH 5.7) was used with Bialaphos (Gold BioTechnology, St.Louis, Mo.). The embryos were transferred to selection media containing3 mg/L Bialaphos until embryogenic isolates were obtained. Recoveredisolates were bulked up by transferring to fresh selection medium at2-week intervals for regeneration and further analysis. Those skilled inthe art of maize transformation will understand that other methods ofselection of transformed plants are available when other plantexpressible selectable marker genes (e.g. herbicide tolerance genes) areused.

Two different constructs of DIG-657 contained in plasmids pDAB114534 andpDAB114535 (SEQ ID NO:5 and SEQ ID NO:6) (Table 5) were transformed intomaize. T0 plants were regenerated and tested for their ability tocontrol target insect pests. Construct pDAB115782 is a plasmidcontaining the gene for a YFP (yellow fluorescent protein) used as anegative control for a plant transformed with a non-insecticidal gene.

TABLE 5 Description of Plasmids containing different DIG- 657 constructstransformed into maize plants. Plasmid: Description: Feature: pDAB114534BorderB::ZmUbi1/IRDIG9931.4/ DIG-657 ZmPer5::ZCZ018::SCBV(MAM)/ fulllength AAD-1/ZmLip::3x Border A (SEQ ID NO: 5) pDAB114535BorderB::ZmUbi1/IRDIG9931.5/ DIG-657 ZmPer5::ZCZ018::SCBV(MAM)/Truncated AAD-1/ZmLip::3x Border A (SEQ ID NO: 6) pDAB115782BorderB::ZmUbi1/PhiYFPv3/ PhiYFP ZmPer5::ZCZ018::SCBV(MAM)/AAD-1/ZmLip::3x Border A

A 1.0×0.5 inch square leaf cutting was taken in duplicate from the V3 orV4 leaf of V5 To transgenic plants. For each test, wild type plant leaftissues were sampled first to prevent cross contamination of Bt toxinson the leaf edges, followed by the transformed plants. In each bioassaytray well (32-well trays and lids, CD International, Pitman, N.J.), 1leaf cutting was placed on 2% water-agar (Fisher Scientific, Fair Lawn,N.J.) and this was replicated 2 times per insect species, per event andper construct. Each well was infested with ten ECB or Diatraeasaccharalis (Fabricius) (Sugarcane Borer “SCB”) neonates (24-48 hrs old)and sealed with a plastic perforated lid to allow for air exchange. Thetrays were placed at 28° C. (16:8 hr light:dark, 40% RH) and after 3days they were graded for percent leaf damage. The percent leaf damagedata was analyzed with ANOVA and mean separations with the Tukey-Kramertest when variances were homogenous by using JMP® Pro 9.0.1 (2010 SASInstitute Inc., Cary, N.C.). The level of leaf damage observed by eachinsect species on transgenic plant material is shown in Table 6.

TABLE 6 Mean percent leaf damage caused by ECB or SCB larvae feeding onT₀ maize leaf tissues from constructs 114534, 114535 or 115782 Average %Leaf Std. Average % Leaf Std. Construct Damage by ECB Dev. Damage by SCBDev. pDAB114534 14.4 15.3 8.7 2.9 (FL DIG-657) pDAB114535 71.8 26.9 93.39.3 (Tr DIG-657) 115782 (YFP) 90.8 8.5 98.8 3.1 * standard deviation (n= 10)

Both constructs 114534 and 114535 resulted in less mean damage ascompared to the YFP negative control construct (115782). Construct114534 expressing the full length DIG-657 protein exhibited greaterinsect activity over construct 114535 expressing the truncated DIG-657protein.

Example 13

Regeneration and seed production.

For regeneration, the cultures were transferred to “28” induction medium(MS salts and vitamins, 30 gm/L sucrose, 5 mg/L Benzylaminopurine, 0.25mg/L 2, 4-D, 3 mg/L Bialaphos, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum,pH 5.7) for 1 week under low-light conditions (14 μEm⁻²s⁻¹) then 1 weekunder high-light conditions (approximately 89 μEm⁻²s⁻¹). Tissues weresubsequently transferred to “36” regeneration medium (same as inductionmedium except lacking plant growth regulators). Plantlets 3-5 cm inlength were transferred to glass culture tubes containing SHGA medium(Schenk and Hildebrandt salts and vitamins (1972); PhytoTechnologyLaboratories., Lenexa, Kans.), 1.0 gm/L myo-inositol, 10 gm/L sucroseand 2.0 gm/L Gellan gum, pH 5.8) to allow for further growth anddevelopment of the shoot and roots. Plants were transplanted to the samesoil mixture as described earlier herein and grown to flowering in thegreenhouse. Controlled pollinations for seed production were conducted.

Example 14

Design of a Plant-Optimized Version of the Coding Sequence for theDIG-657 Bt Insecticidal Protein.

A DNA sequence having a plant codon bias was designed and synthesized toproduce the DIG-657 protein in transgenic monocot and dicot plants. Acodon usage table for maize (Zea mays L.) was calculated from 706protein coding sequences (CDs) obtained from sequences deposited inGenBank. Codon usage tables for tobacco (Nicotiana tabacum, 1268 CDs),canola (Brassica napus, 530 CDs), cotton (Gossypium hirsutum, 197 CDs),and soybean (Glycine max; ca. 1000 CDs) were downloaded from data at thewebsite http://www kazusa.or.jp/codon/. A biased codon set thatcomprises highly used codons common to both maize and dicot datasets, inappropriate weighted average relative amounts, was calculated afteromitting any redundant codon used less than about 10% of total codonuses for that amino acid in either plant type. To derive a plantoptimized sequence encoding the DIG-657 protein, codon substitutions tothe experimentally determined DIG-657 DNA sequence were made such thatthe resulting DNA sequence had the overall codon composition of theplant-optimized codon bias table. Further refinements of the sequencewere made to eliminate undesirable restriction enzyme recognition sites,potential plant intron splice sites, long runs of A/T or C/G residues,and other motifs that might interfere with RNA stability, transcription,or translation of the coding region in plant cells. Other changes weremade to introduce desired restriction enzyme recognition sites, and toeliminate long internal Open Reading Frames (frames other than +1).These changes were all made within the constraints of retaining theplant-biased codon composition. Synthesis of the designed sequence wasperformed by a commercial vendor (DNA2.0, Menlo Park, Calif.).Additional guidance regarding the production of synthetic genes can befound in, for example, WO 97/13402 and U.S. Pat. No. 5,380,831. Amaize-optimized DNA sequence encoding DIG-657 variant1 is given in SEQID NO:3. A Pseudomonas fluorescens optimized DNA sequence encodingDIG-657 variant2 is given in SEQ ID NO:4. A maize-optimized high GC DNAsequence encoding DIG-657 is given in SEQ ID NO:5. A maize-optimizedhigh GC DNA sequence encoding DIG-657 gene truncated 204 aa from theN-terminal is given in SEQ ID NO:6. A dicot-optimized DNA sequenceencoding the full length DIG-657 is disclosed as SEQ ID NO:8. Adicot-optimized DNA sequence encoding the truncated DIG-657 is disclosedas SEQ ID NO:9.

Hybridization of immobilized DNA on Southern blots with radioactivelylabeled gene-specific probes is performed by standard methods Sambrooket al., supra.). Radioactive isotopes used for labeling polynucleotideprobes include 32P, 33P, 14C, or 3H. Incorporation of radioactiveisotopes into polynucleotide probe molecules is done by any of severalmethods well known to those skilled in the field of molecular biology.(See, e.g. Sambrook et al., supra.) In general, hybridization andsubsequent washes are carried out under stringent conditions that allowfor detection of target sequences with homology to the claimed toxinencoding genes. For double-stranded DNA gene probes, hybridization iscarried out overnight at 20° C. to 25° C. below the T_(m) of the DNAhybrid in 6×SSPE, 5×Denhardt's Solution, 0.1% SDS, 0.1 mg/mL denaturedDNA [20×SSPE is 3M NaCl, 0.2 M NaHPO₄, and 0.02M EDTA (ethylenediaminetetra-acetic acid sodium salt); 100×Denhardt's Solution (20 gm/LPolyvinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/L BSA-fractionV).

Washes are typically be carried out as follows:

-   -   a. Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   b. Once at 20° C. below the T_(m) temperature for 15 minutes in        0.2×SSPE, 0.1% SDS (moderate stringency wash).

For oligonucleotide probes, hybridization may be carried out overnightat 10° C. to 20° C. below the T_(m) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/mL denatured DNA. T_(m) foroligonucleotide probes may be determined by the following formula (Suggset al., 1981).T _(m)(° C.)=2(number of T/Abase pairs)+4(number of G/Cbase pairs)

Washes are typically be carried out as follows:

-   -   a. Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   b. Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

Probe molecules for hybridization and hybrid molecules formed betweenprobe and target molecules may be rendered detectable by means otherthan radioactive labeling. Such alternate methods are intended to bewithin the scope of this invention.

While the present disclosure may be susceptible to various modificationsand alternative forms, specific embodiments have been described by wayof example in detail herein. However, it should be understood that thepresent disclosure is not intended to be limited to the particular formsdisclosed. Rather, the present disclosure is to cover all modifications,equivalents, and alternatives falling within the scope of the presentdisclosure as defined by the following appended claims and their legalequivalents.

What is claimed is:
 1. A nucleic acid construct comprising one or moreheterologous regulatory sequences that drives expression of the nucleicacid sequence of SEQ ID NO:9.
 2. A plant, seed, or plant part comprisingthe nucleic acid construct of claim
 1. 3. The plant or plant part ofclaim 2, wherein the polypeptide encoded by the nucleic acid constructhas insecticidal activity against an insect selected from the groupconsisting of European Corn Borer, Cry1F resistant European Corn Borer,Soybean Looper, Velvetbean Caterpillar, Tobacco Budworm, CottonBollworm, Corn Earworm, and Diamondback Moth.
 4. A method for producingan insect resistant plant variety comprising breeding a non-transgenicplant with the transgenic plant of claim 2 and selecting progeny byanalyzing for at least a portion of the foreign DNA construct from saidinsect resistant plant.